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Biomedical subjects

S B Hladky

Publications and source records attributed to S B Hladky.

At least 19 recordsLinked to original sources
PubMed2001

Influences of glutathione on anionic substrate efflux in tumour cells expressing the multidrug resistance-associated protein, MRP1.

The ATP-dependent transport of natural product drugs, e.g. vincristine, by multidrug resistance-associated protein (MRP1) requires reduced glutathione (GSH), whilst that of anionic substrates does not. The present results suggest, however, that GSH can modulate transport of anionic species. Efflux of fluorescent anionic substrates was measured from adherent MRP1-expressing human multidrug-resistant lung tumour cells, COR-L23/R, and drug-sensitive parental cells. As expected, much greater efflux of calcein, methylfluorescein-glutathione (GS-MF), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) was observed from the resistant cells. Unexpectedly, lowering GSH levels in COR-L23/R cells by inhibiting GSH synthesis with buthionine sulfoximine decreased efflux of calcein and of GS-MF (3-fold and 1.6-fold) but not efflux of BCECF. Transport of the anionic conjugate dinitrophenyl-glutathione ([(3)H]DNP-SG) was investigated by following its uptake into inside-out plasma membrane vesicles prepared from the MRP1-expressing cells. At least 90% of the ATP-dependent uptake was blockable by the anti-MRP1 antibody QCRL-3 and 100 microM vincristine inhibited uptake but only in the presence of 1--3 mM GSH, suggesting MRP1 to be the protein primarily responsible for this transport. Agents shown to reduce efflux of calcein from resistant cells, i.e. indomethacin, MK-571, and probenecid, also inhibited [(3)H]DNP-SG uptakes, consistent with MRP1 being responsible for export of calcein. At concentrations achievable within cells, GSSG (70 microM) inhibited uptake whereas GSH (1 and 3 mM) enhanced uptake. We suggest that variations in both GSH and GSSG levels within cells may affect MRP1-mediated anion transport.

ATP-Binding Cassette Transporters↗
PubMed2001

Bayesian restoration of ion channel records using hidden Markov models.

Hidden Markov models have been used to restore recorded signals of single ion channels buried in background noise. Parameter estimation and signal restoration are usually carried out through likelihood maximization by using variants of the Baum-Welch forward-backward procedures. This paper presents an alternative approach for dealing with this inferential task. The inferences are made by using a combination of the framework provided by Bayesian statistics and numerical methods based on Markov chain Monte Carlo stochastic simulation. The reliability of this approach is tested by using synthetic signals of known characteristics. The expectations of the model parameters estimated here are close to those calculated using the Baum-Welch algorithm, but the present methods also yield estimates of their errors. Comparisons of the results of the Bayesian Markov Chain Monte Carlo approach with those obtained by filtering and thresholding demonstrate clearly the superiority of the new methods.

Bayes Theorem↗
PubMed2001

Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells.

Clotrimazole has been shown to have potent anti-malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S-conjugate transporter. This paper shows that transport of the model substrate, 3 microm dinitrophenyl S-glutathione, across erythrocyte membranes is inhibited by multidrug resistance-associated protein 1 (MRP1)-specific antibody, QCRL-3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1-expressing tumour cells, MRP1-associated transport is inhibited by clotrimazole over the range 2-20 microm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1-expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti-malarial action.

Biological Transport↗
PubMed2000

Adjustments for the display of quantized ion channel dwell times in histograms with logarithmic bins.

Dwell-time histograms are often plotted as part of patch-clamp investigations of ion channel currents. The advantages of plotting these histograms with a logarithmic time axis were demonstrated by, J. Physiol. (Lond.). 378:141-174), Pflügers Arch. 410:530-553), and, Biophys. J. 52:1047-1054). Sigworth and Sine argued that the interpretation of such histograms is simplified if the counts are presented in a manner similar to that of a probability density function. However, when ion channel records are recorded as a discrete time series, the dwell times are quantized. As a result, the mapping of dwell times to logarithmically spaced bins is highly irregular; bins may be empty, and significant irregularities may extend beyond the duration of 100 samples. Using simple approximations based on the nature of the binning process and the transformation rules for probability density functions, we develop adjustments for the display of the counts to compensate for this effect. Tests with simulated data suggest that this procedure provides a faithful representation of the data.

Data Interpretation, Statistical↗
PubMed2000

Use of membrane vesicles to investigate drug interactions with transporter proteins, P-glycoprotein and multidrug resistance-associated protein.

BACKGROUND: The ATP-dependent drug transporter proteins, P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) are known to be involved in drug efflux that reduces drug accumulation and so renders tumor cells resistant to the cytotoxic effects of a number of anticancer agents. The ways in which these transporters bring about drug expulsion are not fully explained and may involve intracellular factors as well. Thus detailed evidence may be difficult to obtain from studies on intact cells. MATERIAL AND METHODS: Inside-out plasma membrane vesicles prepared from multidrug-resistant cells expressing high amounts of Pgp or of MRP provide a simpler system for investigating the interactions of putative substrates and resistance modifiers with the transport process. We consider here some aspects of the accumulation of radiolabelled vincristine and of dinitrophenol glutathione conjugate by these vesicles and demonstrate the usefulness of this approach for determining whether potential inhibitors have their effects on transport at the cell membrane or by more indirect means. CONCLUSIONS: We show that information gained from analysis of the ATP-dependence, time course and osmotic sensitivity of accumulation is helpful in distinguishing between transport and changes in binding. We have also used the technique to demonstrate the effects of the resistance modifier, XR-9051 on Pgp-mediated transport and to explore interactions of MK571, indomethacin and ethacrynic acid with MRP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells.

1. Volume-activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole-cell voltage-clamp technique and on a molecular level using the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time-dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I- (1.7) > Br- (1.2) > Cl- (1.0) > F- (0. 7) > gluconate (0.18). 3. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 microM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS, 100 microM) also blocked the hypotonicity-induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume-activated current was also inhibited by the antioestrogen tamoxifen (10 microM). 4. The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5. Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 6. Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 7. Verapamil (100 microM) decreased both the inward and the outward hypotonicity-activated chloride current. 8. RT-PCR analysis was used to determine possible molecular candidates for the volume-sensitive current. Expression of the ClC-2, ClC-3 and ClC-5 chloride channels, as well as pICln, could be shown at the mRNA level. 9. We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC-3 channel currents as described in other cell types.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1999

Can we use rate constants and state models to describe ion transport through gramicidin channels?

Can we use rate constants and state models to describe ion transport through gramicidin channels? Maybe, but only if rate constants are just proportionality constants between rates and probabilities of observing states of the channel. This approach is natural if the system of channel plus ions (plus water) is almost always in one or another of a small number of identifiable states. Many features of ion transport through gramicidin, including the conductance-concentration relationship, concentration-dependent permeability ratios, anomalous mole fraction effect and to some extent flux ratio exponents, are consistent with a description in which there are four occupation 'states' of the pore: only water; an ion at one end; an ion at the other; and ions at both ends. Current-voltage relationships can (and must) also be fitted, but until there is a theory to predict the potential dependence of the rate constants this success will remain hollow. Other features have resisted interpretation. These include the failures to determine 'binding constants' consistent with all the data; the variation of flux ratio exponents with ion type; and, probably, the variation of the currents with asymmetrical ion concentrations. Nevertheless, state models still have one attractive feature, they allow consideration of the effects that one ion within the pore has on the movements of another.

Anti-Bacterial Agents↗
PubMed1998

Streaming potentials in gramicidin channels measured with ion-selective microelectrodes.

Streaming potentials have been measured for gramicidin channels with a new method employing ion-selective microelectrodes. It is shown that ideally ion-selective electrodes placed at the membrane surface record the true streaming potential. Using this method for ion concentrations below 100 mM, approximately seven water molecules are transported whenever a sodium, potassium, or cesium ion, passes through the channel. This new method confirms earlier measurements (Rosenberg, P.A., and A. Finkelstein. 1978. Interaction of ions and water in gramicidin A channels. J. Gen. Physiol. 72:327-340) in which the streaming potentials were calculated as the difference between electrical potentials measured in the presence of gramicidin and in the presence of the ion carriers valinomycin and nonactin.

Anti-Bacterial Agents↗
PubMed1997

Hypotonicity-induced changes in anion permeability of cultured rat brain endothelial cells.

Iodide efflux, an index of anion permeability, has been monitored in cultured rat brain endothelial cells. Following hypotonicity-induced swelling, large, rapid increases in permeability occur, the extent of these increases depending on the degree of hypotonicity. Such large responses are not observed with rat aortic endothelial cells. Results of anion substitution experiments suggest that iodide efflux is via a chloride channel rather than an exchanger. The efflux increase is blocked by NPPB (100 microM) but not by DIDS or DPC at 100 microM. It is dependent on intracellular ATP but unaffected by removal of external calcium. Increasing internal calcium using A23187 does not produce a change in efflux, but depletion of calcium reduces or eliminates the response to hypotonicity. The response is reduced by pimozide (2-50 microM) that inhibits the actions of calmodulin and by pBPB (10 microM) that affects phospholipase A2 activity. It is eliminated by 5-lipoxygenase inhibitors (L-656,224 and ETH615, 10 microM) but is unaffected by cyclo-oxygenase inhibitors (indomethacin and piroxicam, 1-100 microM). It is blocked by some modulators of P-glycoprotein activity, e.g., verapamil (100 microM), tamoxifen (50 microM), and progesterone (100 microM) but not by others, e,g., forskolin (40 microM), dideoxyforskolin (40 microM), quinidine (100 microM) and cyclosporin A (10 microM).

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Hypotonicity-induced anion fluxes in cells expressing the multidrug-resistance-associated protein, MRP.

Anion transport in human multidrug-resistant large cell lung tumour cells (COR-L23/R) which overexpress the multidrug-resistance-associated protein (MRP) has been compared with that in cells of the parent line (COR-L23/P). Whole-cell patch-clamp recordings reveal variability between individual cells in basal anion conductance and in anion conductance increases following exposure to hypotonic media. The increase of stimulated over basal conductance is significantly larger for resistant cells than for parent cells. The chloride channel blocker, diisothiocyanatostilbene-2-2'-disulphonic acid (DIDS), rapidly and reversibly inhibits the increase in outward but not inward conductance when applied externally at 10(-4) M during recording, but it is without effect when introduced into the cells via the patch pipette. Preincubation with DIDS greatly reduces both inward and outward conductance. 125I- efflux has been used to measure anion movement in cell populations. Basal efflux is similar in the two cell lines, but following a hypotonic challenge, the increase in rate constant for efflux from COR-L23/R cells is at least double that from COR-L23/P cells. This increase in efflux is greatly reduced by incubation with DIDS at 10(-4) M. Replacement of external chloride by gluconate does not affect efflux, thus excluding the possible involvement of DIDS-sensitive chloride exchange. Results from both techniques suggest that DIDS-sensitive, hypotonicity-induced anion channel activity is augmented in COR-L23/R multidrug-resistant variant cells which overexpress MRP. This augmentation may be caused by MRP itself or by other genes coexpressed with MRP.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Ion binding constants for gramicidin A obtained from water permeability measurements.

Gramicidin A pores are permeable to water and small monovalent cations. For K, Rb, and Cs there is good evidence from conductances and permeability ratios that a second ion can enter a pore already occupied by another, but for Na this evidence is inconclusive and comparison of tracer fluxes and single channel conductances suggests that second ion entries are prohibited. Partly as a result of the complications of second ion entry there have been widely differing estimates for the dissociation constants for the first ion in the channel. Dani and Levitt (1981, Biophys. J. 35: 485-499) introduced a method for calculating ion binding constants from simultaneous measurements of water fluxes and membrane conductance. They found no evidence for second ion binding and calculated dissociation constants of 115 mM for Li, 69 mM for K, and 2 mM for Tl. It is shown here that the two-ion, four-state model predicts a dependence of water permeability on ion concentration that is difficult to distinguish from the predictions of block by a single ion. Using a modified technique that allows measurement of higher conductances, the first ion dissociation constants have been determined as 80 mM for Na, 40 mM for Rb and 15 mM for Cs. These values and those of Dani and Levitt fall in a smooth sequence. The dissociation constant for Cs is consistent with single channel conductances and flux ratios. There is a discrepancy between this constant for Na and the value, 370 mM, calculated from the single channel conductances and the assumption that a second ion cannot enter or affect an occupied pore. The dissociation constant for Rb is intermediate between those for K and Cs whereas tracer flux measurements (Schagina, Grinfeldt & Lev, 1983. J. Membrane Biol. 73: 203-216) have suggested that Rb interacts much more strongly with the channel than Cs.

Animals↗
PubMed1995

The mechanism of ion conduction by valinomycin: analysis of charge pulse responses.

Even though valinomycin has been employed and studied extensively for over 30 years, the attempts to explain its mechanism have not been entirely successful. The basic carrier model uses four rate constants that describe association of an ion and carrier, transfer of the complex across the membrane, dissociation of the complex, and transfer of the free carrier back across the membrane. If the basic model is correct all of these constants are independent of ion concentration. In previous work with rubidium the rate constants for transfer of free carrier, transfer of complexes, and dissociation were independent of the concentration, but the rate constant for association varied markedly. No satisfactory explanation for these observations was proposed. In this study current relaxations after charge pulses have been analyzed using digital data acquisition, a Bayesian algorithm, and inspection of linear plots of residuals. In agreement with previous results the relaxations for sufficiently high rubidium or potassium concentrations contain three exponential components, but the rate constants for association and dissociation decrease to similar extents as ion concentration increases. A simple extension of the carrier model to allow a more realistic description of association and dissociation is in good agreement with the rate constants fitted in the present study but not those for low ion concentrations found in previous work. At high ion concentrations the rate-limiting step in association appears to be a change in the conformation of the free carrier preceding the bimolecular association reaction. Transfer of neutral, free valinomycin between the surfaces is slower than the transfer of the charged ion-valinomycin complexes. Transfer of the complex may be hastened by deformation of the membrane, or transfer of the free carrier may be slowed by a need for conformation changes.

Algorithms↗
PubMed1994

An upper limit for the effect of low frequency magnetic fields on ATP-sensitive potassium channels.

Currents have been recorded for ATP-sensitive potassium channels in excised patches of membranes from an insulin secreting cell line, CRI-G1. The multi-channel records have been analyzed to reveal the single-channel conductance, the frequency and duration of bursts and the frequency of flickers (with periods between 0.5 and 5 ms). Control records in the absence of applied magnetic fields are similar to those reported by others. Patches have been exposed to parallel static and low frequency magnetic fields including a combination satisfying the 'cyclotron resonance' condition. The fields were applied for 30 s periods interleaved with 30 s controls. No significant differences in channel properties were observed between the control and field exposed periods. The largest change in position of the peak of the distribution of opening and closing transitions was 3%.

Adenosine Triphosphate↗
PubMed1994

Absence of effects of low-frequency, low-amplitude magnetic fields on the properties of gramicidin A channels.

The effects of static and low-frequency magnetic fields on gramicidin A channels have been investigated using bilayer patch clamp recording and a bridge technique capable of detecting 0.3% changes in the conductance of glyceryl monooleate membranes containing many channels. In the bridge technique the conductance was assessed using 10-ms voltage pulses applied at 10 Hz. Measurements were made for LiCl, KCl, and CsCl using magnetic fields of 50, 100, 500, and 5000 microT with the frequency scanned from 10-200 Hz. The combinations of static and low-frequency fields employed include the "cyclotron resonance" conditions at which effects had been predicted to occur. In no case was there any detectable change in conductance when the magnetic fields were applied or changed. Potassium currents through single gramicidin channels have been recorded for patches in which several channels may be open at once. Fields were applied for 2 min periods interleaved with 2 min controls. Methods have been developed to analyze the multichannel records to reveal the amplitude and duration of the channels together with the frequency, depth, and apparent period of flickers. No significant differences were observed between the control and field-exposed recording periods. The peak of the distribution of opening and closing transitions always coincided for fields on and off within the resolution, 0.4%, of the recordings. There are at least two types of flicker, one with typical period less than 0.1 ms, the other with typical period from 0.3-0.8 ms. Most of the latter were not complete closures with the conductance during a flicker 15-20% above the level for a full closure.

Electric Conductivity↗
PubMed1992

Ion channel formation by duramycin.

The formation of ion channels by the nonadecapeptide antibiotic duramycin was examined using black lipid membranes and using the patch-clamp technique. In black lipid membranes made from glyceryl monooleate or a phosphatidylcholine/phosphatidylethanolamine mixture, duramycin induced complex fluctuations in membrane conductance, some step-like and some which were incapable of being resolved into discrete conductance states. Both conductance and largest step size increased with time. A similar time-dependent increase in conductance was seen in patch-clamp experiments with HCA-7 Colony 29 human colonic epithelial cell. The channels displayed weak anion selectivity and the smaller channels formed in patches from epithelial cells showed weak inward-rectification. Channel formation by duramycin was achieved at lower concentrations when the black lipid membrane was made with phospholipid rather than with glyceryl monooleate. Lower concentrations were effective in generating conductances in epithelial cells than in bilayers. It is concluded that duramycin forms ion channels in both artificial and biological membranes. Accumulation of duramycin and coalescence of initially small channels into larger ones is considered to be responsible for the recorded behaviour and to final disruption of membranes.

Anti-Bacterial Agents↗
PubMed1991

Stimulation of sodium transport by duramycin in cultured human colonic epithelia.

Effects of two peptide antibiotics, duramycin and Ro 09-0198, on ion transport in cultured human colonic epithelia were investigated. Both peptides acted on the apical face of epithelial monolayers, causing an increase in ion transport measured as short circuit current. Concentration-response relationships were complex, because above a concentration of 2 to 5 microM, the peptides caused currents either to decline toward zero or become large and unstable. Ion substitution experiments showed that the majority of the current response was due to electrogenic sodium absorption. An outward chloride current could also be induced by duramycin, provided an outwardly directed chloride gradient was imposed. It was also shown that the peptides could increase [Ca]i, probably by creating entry sites in the apical face. Duramycin was also able to create large conductance (2000 pS) channels in "black" lipid bilayers. It is proposed that the antibiotics interact with membrane lipids in the apical faces of colonic epithelia to create artificial nonspecific ion channels. The prevailing electrochemical gradients which exist when the epithelia are bathed symmetrically in Krebs-Henseleit solution result in increased electrogenic sodium transport. No evidence was found for colonic epithelia that duramycin or Ro 09-0198 interacted specifically with either membrane channels or receptors to increase ion transport.

Amino Acid Sequence↗